19 research outputs found
Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vκ Gene Rearrangement in Pre-B Cells and Pro-B Cells
To date there has not been a study directly comparing relative Igκ rearrangement frequencies obtained from genomic DNA (gDNA) and cDNA and since each approach has potential biases, this is an important issue to clarify. Here we used deep sequencing to compare the unbiased gDNA and RNA Igκ repertoire from the same pre-B cell pool. We find that ~20% of Vκ genes have rearrangement frequencies ≥2-fold up or down in RNA vs. DNA libraries, including many members of the Vκ3, Vκ4, and Vκ6 families. Regression analysis indicates Ikaros and E2A binding are associated with strong promoters. Within the pre-B cell repertoire, we observed that individual Vκ genes rearranged at very different frequencies, and also displayed very different Jκ usage. Regression analysis revealed that the greatly unequal Vκ gene rearrangement frequencies are best predicted by epigenetic marks of enhancers. In particular, the levels of newly arising H3K4me1 peaks associated with many Vκ genes in pre-B cells are most predictive of rearrangement levels. Since H3K4me1 is associated with long range chromatin interactions which are created during locus contraction, our data provides mechanistic insight into unequal rearrangement levels. Comparison of Igκ rearrangements occurring in pro-B cells and pre-B cells from the same mice reveal a pro-B cell bias toward usage of Jκ-distal Vκ genes, particularly Vκ10-96 and Vκ1-135. Regression analysis indicates that PU.1 binding is the highest predictor of Vκ gene rearrangement frequency in pro-B cells. Lastly, the repertoires of iEκ−/− pre-B cells reveal that iEκ actively influences Vκ gene usage, particularly Vκ3 family genes, overlapping with a zone of iEκ-regulated germline transcription. These represent new roles for iEκ in addition to its critical function in promoting overall Igκ rearrangement. Together, this study provides insight into many aspects of Igκ repertoire formation
Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance
Chemical synapses contain multitudes of proteins, which in common with all proteins, have finite lifetimes and therefore need to be continuously replaced. Given the huge numbers of synaptic connections typical neurons form, the demand to maintain the protein contents of these connections might be expected to place considerable metabolic demands on each neuron. Moreover, synaptic proteostasis might differ according to distance from global protein synthesis sites, the availability of distributed protein synthesis facilities, trafficking rates and synaptic protein dynamics. To date, the turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus metabolic demands or the aforementioned relationships remain largely unknown. In the current study we used dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), Fluorescent Non-Canonical Amino acid Tagging (FUNCAT), quantitative immunohistochemistry and bioinformatics to systematically measure the metabolic half-lives of hundreds of synaptic proteins, examine how these depend on their pre/postsynaptic affiliation or their association with particular molecular complexes, and assess the metabolic load of synaptic proteostasis. We found that nearly all synaptic proteins identified here exhibited half-lifetimes in the range of 2-5 days. Unexpectedly, metabolic turnover rates were not significantly different for presynaptic and postsynaptic proteins, or for proteins for which mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited very similar turnover rates, indicating that their biogenesis and degradation might be coupled, a possibility further supported by bioinformatics-based analyses. The relatively low turnover rates measured here (∼0.7% of synaptic protein content per hour) are in good agreement with imaging-based studies of synaptic protein trafficking, yet indicate that the metabolic load synaptic protein turnover places on individual neurons is very substantial
Co-Transcriptional Splicing and Functional Role of PKCβ in Insulin-Sensitive L6 Skeletal Muscle Cells and 3T3-L1 Adipocytes
PKC
βII is alternatively spliced during acute insulin stimulation in L6 skeletal
muscle cells. This PKC
βII isoform is critical in propagating GLUT4 translocation.
PKC
β protein and promoter dysfunction correlate with human insulin resistance. TZD
treatment ameliorates whole-body insulin-resistance. Its primary target is adipocyte
PPAR
γ, which it activates upon binding. This causes both altered circulating serum FFA
concentrations and adipokine secretion profile. How TZDs affect the intracellular
signaling of skeletal muscle cells is unknown. RT-PCR and Western blot analysis
showed that TZDs elevated PKC
βII by a process that involves co-transcriptional splicing.
PGC1
α overexpression most closely resembled TZD treatment by increasing PKCβII
protein levels and keeping PKC
βI levels relatively constant. Use of a heterologous PKCβ
promoter driven PKC
β minigene demonstrated that PPARγ could regulate the PKCβ
promoter, but whether this is direct or indirect is unclear. SRp40 splicing factor has been
shown to dock onto the PGC1
α CTD and influence splicing. SRp40, through
overexpression and silencing, appears to play a part in PKC
β promoter regulation.
PKC β promoter regulation was also studied in 3T3-L1 cells. TZDs were
experimentally shown to have no role in PKC β promoter regulation despite PPARγ
activation. Chromatin immunoprecipitation assays revealed PU.1 as a putative PKC β
transcription factor that can cross-talk with the spliceosome, possibly through SRp40
which was also associated with the PKC β
promoter. 3T3-L1 adipocyte differentiation
revealed a novel developmentally-regulated switch from PKC βI to PKCβ
II, using
western blot and Real-Time PCR analysis. Pharmacological inhibition of PKC β
II using
CGP53353 and LY379196 blocked [ 3
H]2-deoxyglucose uptake and revealed a functional
role for PKC β
II in adipocyte ISGT. CGP53353 specifically inhibited phosphorylation of
PKC β
II Serine 660 and not other critical upstream components of the insulin signaling
pathway. Subcellular fractionation and PM sheet assay pointed to PKC β
II-mediated
regulation of GLUT4 translocation to the PM. Co-immunoprecipitation between PKC β
II
and GLUT4 allude to possible direct interaction. Western blot and immunofluorescence
assays show PKC β
II activity is linked with Akt Serine 473 phosphorylation, thus full Akt
activity. Western blot and co-immunoprecipitation suggested that insulin caused active
mTORC2 to directly activate PKC βII. Data support a model whereby PKCβ
II is
downstream of mTORC2 yet upstream of Akt, thereby regulating GLUT4 translocation
Cutting Edge: Proper Orientation of CTCF Sites in Cer Is Required for Normal Jκ-Distal and Jκ-Proximal Vκ Gene Usage
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Induction and Potential Reversal of a T Cell Exhaustion-Like State: In Vitro Potency Assay for Functional Screening of Immune Checkpoint Drug Candidates
Tumor infiltrating lymphocytes (TIL) entering the tumor microenvironment (TME) encounter many suppressive factors resulting in a spectrum of possible differentiation paths, many with overlapping characteristics. This differentiation spectrum includes T cell anergy, unresponsiveness, quiescence, tolerance, dysfunction/suppression, and exhaustion, each with many subtypes. Immune checkpoint blockade (ICB) cancer drug therapies have focused on reinvigorating exhausted T cells (T-EX) and dysfunctional T cells (herein referred to as TEX). Many factors have been attributed to as causative to this exhausted state including sustained surface expression of multiple co-inhibitory receptors, altered transcription factor expression, epigenetic rewiring, and dysregulated metabolism. Antigen persistence is necessary for driving TEX maintenance in both the chronic viral infection setting and cancer. In addition to persistent antigen exposure, TILs within the TME encounter numerous tumor-mediated immunosuppressive metabolic by-products, suppressive cytokines, hypoxia, and cellular debris which converge to suppress T cell function and uniquely alter its transcription factor profile. These suppressed or dysfunctional T cells are incapable of mounting an optimal anti-tumor response in part due to lack of fitness in competing for glucose and oxygen. This chapter describes two different potency assays: (a) first we describe optimization of a core recall antigen-based in vitro potency assay for screening of immunopotentiating drug candidates; (b) the second assay bundles the core assay with further addition of immunosuppressive agents derived from the TME making a customizable T cell exhaustion-like assay. Our recall antigen assay is being used as a tool for function-based screening of ICB drug candidates. In this assay healthy human Peripheral Blood Mononuclear Cells (PBMCs) are stimulated with peptide(s) and grown in culture for 1 week. Day 4 supernatants are functionally assayed by ELISA for IFN-gamma secretion, and cells are assayed on day 7 by flow cytometry for CD8(+) or CD4(+) T cell expansion by using a single or cocktail of pMHC tetramers. We have shown that in roughly 30% of donor PBMCs, ICB drugs such as pembrolizumab are able to boost both IFN-gamma secretion and antigen-specific recall. One potential explanation for this effect is that the observed increase in T cell co-inhibitory receptor expression and presumed co-inhibitory receptor downstream signaling is ameliorated with ICB drugs releasing these T cells from the repressive effects of these co-inhibitory receptors. We have further enhanced our recall assay to more closely resemble the TME by including metabolites and other suppressive agents at concentrations not normally encountered by T cells in a healthy environment. We show one example of this whereby addition of adenosine to the culture results in suppression of antigen-specific T cell expansion without affecting cell viability. Further, we screened several drug candidates on their ability to counteract the negative effects of adenosine and found that one of the drugs was indeed able to restore antigen-specific T cell expansion. Hence, this TME-based recall antigen assay allows for dissection of the effects of TME-based factors on donor-specific T cell response as well as drug candidate screening
Critical Role of B Cell Lymphoma 10 in BAFF-Regulated NF-κB Activation and Survival of Anergic B Cells
Anergy is a key physiological mechanism for restraining self-reactive B cells. A marked portion of peripheral B cells are anergic B cells that largely depend on B cell-activating factor (BAFF) for survival. BAFF activates the canonical and noncanonical NF-κB pathways, both of which are required for B cell survival. Here we report that deficiency of the adaptor protein B cell lymphoma 10 (Bcl10) impaired the ability of BAFF to support B cell survival in vitro, and specifically increased apoptosis in anergic B cells in vivo, dramatically reducing anergic B cells in mice. Bcl10-dependent survival of self-reactive anergic B cells was confirmed in the Ig(HEL)sHEL double-transgenic mouse model of B cell anergy. Further, we found that BAFF stimulation induced Bcl10 association with IKKβ, a key component of the canonical NF-κB pathway. Consistently, Bcl10-deficient B cells were impaired in BAFF-induced IκBα phosphorylation and formation of nuclear p50:c-Rel complexes. Bcl10-deficient B cells also displayed reduced expression of NF-κB2/p100, severely reducing BAFF-induced nuclear accumulation of noncanonical p52:RelB complexes. Consequently, Bcl10-deficient B cells failed to express Bcl-xL, a BAFF-induced NF-κB target gene. Taken together, these data demonstrate that Bcl10 controls BAFF-induced canonical NF-κB activation directly and noncanonical NF-κB activation indirectly. The BAFF-R/Bcl10/NF-κB signaling axis plays a critical role in peripheral B cell tolerance by regulating the survival of self-reactive anergic B cells
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Defining the mechanisms of B cell initiated autoimmune disease
Abstract Systemic lupus erythematosus (SLE) afflicts more than 1.5 million individuals in the United States with very limited and debilitating therapeutic options. The etiology of the disease remains unclear. It is hypothesized that in SLE, peripheral B cell tolerance is breached by inappropriate survival of autoreactive cells. Consistently, overexpression of B cell activating factor (BAFF), a key regulator of B cell survival can rescue autoreactive B cells and contribute to autoimmune disease. BAFF counters apoptosis in part through decreasing BH3-only pro-apoptotic protein Bim. Notably, both BAFF and Bim are physiological regulators of B cell tolerance. Therefore, to define the role of B cells and B cell tolerance in the initiation and progression of autoimmune disease we created a mouse model wherein gene encoding Bim is selectively deleted in B cells (B.Bimf/f). Initial analyses of these mice suggest that B cells can initiate an autoimmune pathogenesis phenotypically similar to SLE, with mice exhibiting splenomegaly and increased CD21loCD23lo B cells. Given that persistent activity of NF-kB in B cells can also result in autoimmune diseases, we are now investigating whether the persistent NF-kB and dysregulated apoptosis can synergize in SLE pathogenesis. To that end, we have crossed B.Bimf/f mice with a mouse line that lacks a recently discovered potential negative regulator of NF-kB, ZFAND6 (B.Bimf/f x ZfanD6−/−). Preliminary findings suggest that the compound mutant mice display exacerbated autoimmune symptoms with higher incidence of splenomegaly and kidney pathology. These preliminary results suggest that a breach in tolerance (B.Bimf/f ) and persistent NF-kB activity cooperate in the initiation and severity of SLE pathogenesis
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Immunoglobulin gene rearrangement and BAFF responsive maturation defines a novel B cell population undergoing extra-BM development
Abstract Transitional type-1 (T1) cells are peripheral immature B cells known to populate the spleen (spl) after completing their BCR assembly in the bone marrow (BM). To advance the understanding of splenic T1 (CD19posCD24hiCD21neg) B cells, we addressed the heterogeneity and biology of these cells using flow cytometry combined with genetically modified mice. Most recent emigrant T1 cells were selected by excluding CD23pos and including CD93high (AA4.1) B cells termed T12123DN. Transcriptomic analysis identified RAG1 and 2 as signature genes for this B cell population. Further separation of T12123DN cells based on surface IgM expression revealed a previously undescribed cell subset with undetectable cell surface IgM (-IgMneg). The spl-IgMneg subsets expresses RAG1/2 and actively undergoes Igk gene rearrangement at levels comparable to BM pre-B cells. Upon in vitro exposure to BAFF or transplantation into immunodeficient hosts, spl-IgMneg cells can give rise to fully mature IgMposIgDpos B cells. Furthermore, BAFF-R and NF-kB pathways are required for their efficient maturation. Our findings suggest that the spl-T1 population encompasses a subset of B cells that resemble but are distinct from the developing B cells in the BM. These spl-IgMneg B cells may represent receptor editing B cells, and/or precursor B cells undergoing BCR assembly and selection in the periphery, possibly providing an opportunity for tolerance induction to tissue restricted self-antigens and microbiota-derived antigens
Impaired B Cell Apoptosis Results in Autoimmunity That Is Alleviated by Ablation of Btk
While apoptosis plays a role in B-cell self-tolerance, its significance in preventing autoimmunity remains unclear. Here, we report that dysregulated B cell apoptosis leads to delayed onset autoimmune phenotype in mice. Our longitudinal studies revealed that mice with B cell-specific deletion of pro-apoptotic Bim (
) have an expanded B cell compartment with a notable increase in transitional, antibody secreting and recently described double negative (DN) B cells. They develop greater hypergammaglobulinemia than mice lacking Bim in all cells and accumulate several autoantibodies characteristic of Systemic Lupus Erythematosus (SLE) and related Sjögren's Syndrome (SS) including anti-nuclear, anti-Ro/SSA and anti-La/SSB at a level comparable to NODH2h4 autoimmune mouse model. Furthermore, lymphocytes infiltrated the tissues including submandibular glands and formed follicle-like structures populated with B cells, plasma cells and T follicular helper cells indicative of ongoing immune reaction. This autoimmunity was ameliorated upon deletion of Bruton's tyrosine kinase (Btk) gene, which encodes a key B cell signaling protein. These studies suggest that Bim-mediated apoptosis suppresses and B cell tyrosine kinase signaling promotes B cell-mediated autoimmunity